Integrins and the proinflammatory phospholipase A2

描述（由申请人提供）：分泌型磷脂酶A2组MA （sPLA2-IIA）是PLA2家族成员之一，是一种急性期反应物，其血浆水平在炎症细胞因子的全身性炎症条件下显着升高。进一步的循环sPLA2-IIA水平是冠状动脉疾病的独立危险标志，并预测心血管事件。SPLA2-IIA诱导单核细胞、角质形成细胞和内皮细胞的增殖、分化、细胞因子产生和核因子(NF)-kB活化。这种酶水解存在于脂蛋白和细胞膜中的甘油酰磷脂中的sn-2酯键，形成非酯化脂肪酸（如花生四烯酸）和溶血磷脂，它们作为细胞内的第二信使或进一步代谢为有效的炎症介质。事实上，表达人sPLA2-IIA的转基因小鼠具有角化细胞过度增殖和更多的动脉粥样硬化。然而，sPLA2-IIA的一些生物学效应是独立于其催化功能的，表明它们是通过sPLA2-IIA与特定受体结合而介导的。在小鼠中鉴定出sPLA2-IIA的高亲和力受体（m型受体）。然而，人sPLA2-IIA不与人m型受体结合，人sPLA2-IIA受体尚不清楚。我们最近发现人类SPLA2-IIA特异性结合整合素α - β。我们通过分子对接模拟和定点诱变产生了不与整合素结合的sPLA2突变体。我们假设1)整合素作为sPLA2-IIA的受体，2)整合素通过整合素途径转导sPLA2-IIA的促炎细胞内信号，3)阻断sPLA2-IIA-整合素相互作用阻断sPLA2-IIA的促炎功能。整合素结合缺陷sPLA2-IIA突变体是研究整合素在sPLA2-IIA信号传导中的作用的有力工具。为了解决这一假设，我们提出1)表征SPLA2-IIA-整合素的相互作用，并鉴定在不同细胞类型（如单核细胞）中特异性结合SPLA2-IIA的整合素。2)利用整合素结合的sPLA2缺陷突变体在不同类型的炎症相关细胞中鉴定整合素介导的sPLA2 - iia胞内信号。本实验将确定整合素在单核细胞和内皮细胞sPLA2-IIA的信号传导和促炎活性中的作用，并将整合素-sPLA2-IIA相互作用作为炎症性疾病的新治疗靶点。

DESCRIPTION (provided by applicant): The secretory phospholipase A2 group MA (sPLA2-IIA), one of the PLA2 family members, is an acute-phase reactant and its plasma levels are markedly increased during systemic inflammatory conditions by inflammatory cytokines. Further circulating sPLA2-IIA levels constitute an independent risk marker for coronary artery disease and predict cardiovascular events. SPLA2-IIA induces proliferation, differentiation, and cytokine production, and nuclear factor (NF)-kB activation in monocytes, keratinocytes, and endothelial cells. This enzyme hydrolyzes the sn-2 ester bond in the glyceroacyl phospholipids present in lipoproteins and cell membranes, forming nonesterified fatty acids (e.g., arachidonic acid) and lysophospholipids, which act as intracellular second messengers or are further metabolized into potent mediators for inflammation. Indeed, transgenic mice expressing human sPLA2-IIA have hyperproliferation of keratinocytes and more atherosclerosis. However, some biological effects of sPLA2-IIA are independent of its catalytic function, suggesting that they are mediated by sPLA2-IIA binding to specific receptors. A high-affinity receptor for sPLA2-IIA (the M-type receptor) was identified in mice. However, the human sPLA2-IIA does not bind to the human M-type receptor, and the sPLA2-IIA receptor in human was unknown. We recently discovered that human SPLA2-IIA specifically binds to integrin alphaVbetaS. We generated sPLA2 mutants that do not bind to integrins by molecular docking simulation and site-directed mutagenesis. We hypothesize that 1) integrins serve as receptors for sPLA2-IIA, 2) integrins transduce pro-inflammatory intracellular signals from sPLA2- IIA through integrin pathways, and 3) blocking sPLA2-IIA-integrin interaction blocks pro-inflammatory functions of SPLA2-IIA. The integrin-binding defective sPLA2-IIA mutants are powerful tool to study the role of integrins in sPLA2-IIA signaling. To address this hypothesis, we propose to 1) characterize the SPLA2-IIA- integrin interaction and identify integrins that specifically bind to sPLA2-IIA in different cell types (e.g., monocytes). 2) Identify the integrin-mediated SPLA2-IIA intracellular signals using integrin-binding defective sPLA2 mutants in different cell types involved in inflammation. The proposed experiments will determine the roles of integrins in signaling and pro-inflammatory activity of sPLA2-IIA in monocytes and endothelial cells, and will establish integrin-sPLA2-IIA interaction as a novel therapeutic target for inflammatory diseases.